Role of Membrane Lipid Rafts in MRP4 (ABCC4) Dependent Regulation of the cAMP Pathway in Blood Platelets.

BACKGROUND: Platelet cytosolic cyclic adenosine monophosphate (cAMP) levels are balanced by synthesis, degradation, and efflux. Efflux can occur via multidrug resistant protein-4 (MRP4; ABCC4) present on dense granule and/or plasma membranes. As lipid rafts have been shown to interfere on cAMP homeostasis, we evaluated the relationships between the distribution and activity of MRP4 in lipid rafts and cAMP efflux. METHODS: Platelet activation and cAMP homeostasis were analyzed in human and wild-type or MRP4-deleted mouse platelets in the presence of methyl-ß-cyclodextrin (MßCD) to disrupt lipid rafts, and of activators of the cAMP signalling pathways. Human platelet MRP4 and effector proteins of the cAMP pathway were analyzed by immunoblots in lipid rafts isolated by differential centrifugation. RESULTS: MßCD dose dependently inhibited human and mouse platelet aggregation without affecting per se cAMP levels. An additive inhibitory effect existed between the adenylate cyclase (AC) activator forskolin and MßCD that was accompanied by an overincrease of cAMP, and which was significantly enhanced upon MRP4 deletion. Finally, an efflux of cAMP out of resting platelets incubated with prostaglandin E1 (PGE1) was observed that was partly dependent on MRP4. Lipid rafts contained a small fraction (≈15%) of MRP4 and most of the inhibitory G-protein Gi, whereas Gs protein, AC3, and phosphodiesterases PDE2 and PDE3A were all present as only trace amounts. CONCLUSION: Our results are in favour of part of MRP4 present at the platelet surface, including in lipid rafts. Lipid raft integrity is necessary for cAMP signalling regulation, although MRP4 and most players of cAMP homeostasis are essentially located outside rafts.

Efficacy of pentoxifylline-tocopherol-clodronate in mandibular osteoradionecrosis.

OBJECTIVES/HYPOTHESIS: PENTOCLO treatment, associating pentoxifylline, tocopherol, and clodronate, resolves radiation-induced fibrosis. The main aim of the present study was to prospectively assess efficacy in mandibular osteoradionecrosis (ORN). STUDY DESIGN: Prospective cohort study. METHODS: Twenty-seven patients with mandibular ORN were included in the Pentoclauvergne Study between January 2014 and February 2016. After an initial 28-day phase of antibiotic, antifungal, and corticosteroid therapy, they received the PENTOCLO association daily until cure or a maximum of 24 months. The main assessment criterion was exposed bone area (EBA); secondary criteria comprised the Subjective, objective, management, and analytic (SOMA) score. RESULTS: Under PENTOCLO, EBA decreased by 28% at 2 months, 55% at 6 months, and 92% at 24 months; the SOMA score decreased by 23%, 38%, and 50%, respectively. A complete treatment course cured 76.5% of patients at a mean 9.6 months. CONCLUSIONS: PENTOCLO is a simple, well-tolerated, and effective treatment for mandibular ORN. LEVEL OF EVIDENCE: 4 Laryngoscope, 130:E559-E566, 2020.

Evaluation of commonly used tests to measure the effect of single-dose aspirin on mouse hemostasis.

Discrepancies in preclinical studies of aspirin (ASA) antiplatelet activity in mouse models of bleeding and arterial thrombosis led us to evaluate commonly reported methods in order to propose a procedure for reliably measuring the effects of single dose ASA on mouse hemostasis. FVB and C57Bl6 mice received 100 mg/kg of ASA or vehicle orally 30 min or 3 h prior to investigate either hemostasis using the tail bleeding assay or carotid thrombosis induced by FeCl3, or to blood sampling for isolated platelet aggregation and TXB2 generation. Expected inhibition of COX1 by ASA was ascertained by a strong decrease in TXB2 production, and its effect on platelet function and hemostasis, by decreased collagen-induced aggregation and increased bleeding time, respectively. Strikingly, we determined that anti-hemostatic effects of ASA were more predictable 30 min after administration than 3 h later. Conversely, ASA did not alter time to arterial occlusion of the carotid upon FeCl3-induced thrombosis, suggesting ASA not to be used as reference inhibitor drug in this model of arterial thrombosis.

Low-Dose and Long-Term Olaparib Treatment Sensitizes MDA-MB-231 and SUM1315 Triple-Negative Breast Cancers Spheroids to Fractioned Radiotherapy.

The Triple-Negative Breast Cancer subtype (TNBC) is particularly aggressive and heterogeneous. Thus, Poly-ADP-Ribose Polymerase inhibitors were developed to improve the prognosis of patients and treatment protocols are still being evaluated. In this context, we modelized the efficacy of Olaparib (i.e., 5 and 50 µM), combined with fractioned irradiation (i.e., 5 × 2 Gy) on two aggressive TNBC cell lines MDA-MB-231 (BRCAness) and SUM1315 (BRCA1-mutated). In 2D cell culture and for both models, the clonogenicity drop was 95-fold higher after 5 µM Olaparib and 10 Gy irradiation than Olaparib treatment alone and was only 2-fold higher after 50 µM and 10 Gy. Similar responses were obtained on TNBC tumor-like spheroid models after 10 days of co-treatment. Indeed, the ratio of metabolic activity decrease was of 1.2 for SUM1315 and 3.3 for MDA-MB-231 after 5 µM and 10 Gy and of only 0.9 (both models) after 50 µM and 10 Gy. MDA-MB-231, exhibiting a strong proliferation profile and an overexpression of AURKA, was more sensitive to the co-treatment than SUM1315 cell line, with a stem-cell like phenotype. These results suggest that, with the studied models, the potentiation of Olaparib treatment could be reached with low-dose and long-term exposure combined with fractioned irradiation.

Postoperative volumetric modulated arc therapy for sinonasal cancer: Improved survival compared with 3D conformal radiation therapy.

BACKGROUND: Prospective evaluation of the results of volumetric modulated arc therapy (VMAT) for sinonasal cancer compared to 3D conformal radiation therapy (3DCRT). MATERIALS AND METHODS: We prospectively evaluated 34 patients (pts) treated with postoperative VMAT with simultaneous integrated boost (SIB; RapidArc) from 2011 to 2015. These pts were retrospectively compared with 24 pts treated with 3DCRT from 2003 to 2011. The two sets were not significantly different on sex, mean age, tumor site, stage, histology. Efficacy and toxicity were evaluated. RESULTS: Median follow-up was 45 months (range: 6-143 months). Three-year overall survival was 85.2% in VMAT-SIB versus 65.2% in 3DCRT (P = .02). Three-year local control was 81.2% in VMAT-SIB versus 62.5% in 3DCRT (P = .04). There was a reduction of acute (<0.09) and late (0.03) ocular toxicity of grade ≥ 2 for pts with VMAT-SIB. CONCLUSION: VMAT significantly improved local control and overall survival in sinonasal cancer with lower rate of toxicity.

Fractionated stereotactic radiotherapy of benign skull-base tumors: a dosimetric comparison of volumetric modulated arc therapy with Rapidarc® versus non-coplanar dynamic arcs.

BACKGROUND: Benign tumors of the skull base are a challenge when delivering radiotherapy. An appropriate choice of radiation technique may significantly improve the patient's outcomes. Our study aimed to compare the dosimetric results of fractionated stereotactic radiotherapy between non-coplanar dynamic arcs and coplanar volumetric modulated arctherapy (Rapidarc®). METHODS: Thirteen patients treated with Novalis TX® were analysed: six vestibular schwannomas, four pituitary adenomas and three meningioma. Two treatment plans were created for each case: dynamic arcs (4-5 non coplanar arcs) and Rapidarc® (2 coplanar arcs). All tumors were >3 cm and accessible to both techniques. Patients had a stereotactic facemask (Brainlab) and were daily repositioned by Exactrac®. GTV and CTV were contoured according to tumor type. A 1-mm margin was added to the CTV to obtain PTV. Radiation doses were 52.2-54 Gy, using 1.8 Gy per fraction. Treatment time was faster with Rapidarc®. RESULTS: The mean PTV V95 % was 98.8 for Rapidarc® and 95.9 % for DA (p = 0.09). Homogeneity index was better with Rapidarc®: 0.06 vs. 0.09 (p = 0.01). Higher conformity index values were obtained with Rapidarc®: 75.2 vs. 67.9 % (p = 0.04). The volume of healthy brain that received a high dose (V90 %) was 0.7 % using Rapidarc® vs. 1.4 % with dynamic arcs (p = 0.05). Rapidarc® and dynamic arcs gave, respectively, a mean D40 % of 10.5 vs. 18.1 Gy (p = 0.005) for the hippocampus and a Dmean of 25.4 vs. 35.3 Gy (p = 0.008) for the ipsilateral cochlea. Low-dose delivery with Rapidarc® and dynamic arcs were, respectively, 184 vs. 166 cm(3) for V20 Gy (p = 0.14) and 1265 vs. 1056 cm(3) for V5 Gy (p = 0.67). CONCLUSIONS: Fractionated stereotactic radiotherapy using Rapidarc® for large benign tumors of the skull base provided target volume coverage that was at least equal to that of dynamics arcs, with better conformity and homogeneity and faster treatment time. Rapidarc® also offered better sparing of the ipsilateral cochlea and hippocampus. Low-dose delivery were similar between both techniques.